Environmental exposures, like those occurring at Superfund sites and Brownfields in Rhode Island, nvolve complex mixtures of hazardous chemicals. We have established a novel co-exposure paradigm to expand our mechanistic understanding of testicular injury resulting from such complex exposures. Model esticular toxicants are used as functional probes of the interacting cell types within the seminiferous epithelium: 2,5-hexanedione, carbendazim, and mono-(2-ethylhexyl) phthalate target Sertoli cells, and xrradiation and 1,2-dibromo-3-chloropropane target germ cells. Using the adult rat as the animal model, the co-exposure paradigm combines subacute exposure to 2,5-hexanedione with acute exposure to another Sertoli cell or germ cell toxicant. In the initial funding period, dose-response behavior and phenotypic alterations were determined for the testicular toxicants. Compared with acute toxicant exposure alone, the co-exposure paradigm attenuated or enhanced the germ cell apoptotic response, the final common pathway of testicular injury, depending on dose and cellular target. Strikingly, the gene array analysis supports a positive correlation between a muted or exaggerated gene expression response and the extent of co-exposure attenuation or enhancement of germ cell apoptosis. These exciting results provide a phenotypic anchor for further molecular analyses, and underscore the ability of the co-exposure paradigm to provide new insight into the testicular response to complex exposures. In the next funding period, laser capture microdissection will be used for cell-type and stage-specific enrichment of mRNA and protein from the seminiferous epithelium, extending the co-exposure paradigm to low doses. In addition, novel sperm biomarkers of effect will be identified for chronic cell-type specific testicular injury. The work will be guided by the following working hypotheses: 1) co-exposure attenuation or enhancement of toxicity depends upon dose, targeting, and the extent of molecular perturbation, and 2) the testicular response to cell type specific toxicant exposure can be identified through molecular analysis of sperm. This project contributes to the Molecular Epidemiology & Reproduction Interdisciplinary Focus Area, and identifies principles of paracrine-dependent co-exposure-induced toxicity that are broadly applicable to many organ systems.